N-terminal transmembrane domain of lipase LipA from Pseudomonas protegens Pf-5: A must for its efficient folding into an active conformation

作者：Zha, Daiming; Zhang, Huaidong; Zhang, Houjin; Xu, Li; Yan, Yunjun^*

来源：Biochimie, 2014, 105: 165-171.

DOI：10.1016/j.biochi.2014.07.007

• Pseudomonas protegens lipase
• Transmembrane domain
• Intramolecular chaperone
• 3D structure

LipA from Pseudomonas protegens Pf-5 has been proven not to be secreted into the extracytoplasmic space, proposing that it is a membrane protein in virtue of its N-terminal transmembrane domain predicted by the TMHMM 2.0. However, LipA was confirmed to be an intracellular protein through determining the effects of lipA deletion or overexpression on the lipase activities in the whole-cell, lysis supernatant and lysis pellet, even through its transmembrane domain being able to make heterologous LacZ locate on the cytoplasmic membrane via construction of beta-galactosidase reporter strains. Subsequently, lipase activity assays showed that the transmembrane domain played an indispensable role for the catalytic function of LipA through construction of the markerless deletion mutant of transmembrane domain sequence of lipA and the expression and purification of LipA and LipA Delta TMD. To further investigate why the transmembrane domain lost its membrane localization function and significantly affected the catalytic function of LipA, the 3D structures of LipA and LipA Delta TMD were constructed. The results indicated that the transmembrane domain, located in the interior of LipA, helped the alpha-helical lid to form an open conformation by the mediation of alpha 5 helix. It seems to act as a kind of intramolecular chaperone like the beta-roll motif of subfamily 1.3 lipases, which is novel and is the first to notify the intramolecular chaperone of a subfamily 1.1 lipase.

• 出版日期2014-10
• 单位华中科技大学